Method for measuring the concentration of protease inhibitors, kit for use in such a method and method for dissolving a substrate

Abstract

This invention provides a method for measuring the concentration of urinary trypsin inhibitors which is excellent in precision and reproducibility, whose operation is simple, and in which a possibility of damaging a plastic cell is eliminated. The method for measuring the concentration of urinary trypsin inhibitors comprises mixing an urine sample, a protease solution containing trypsin, and a buffer solution, adding a substrate solution to the mixture to cause the enzyme reaction, and measuring the activity of the enzyme, wherein the buffer solution is prepared so that it contains at least 0.15 mu mol calcium per 1 mu g of the trypsin but no more than 100 mu mol calcium per 1 ml of the urine sample in the reaction mixture, and wherein the substrate solution is prepared by dissolving the substrate in an organic solvent and diluting the mixture solution with aqueous medium, wherein at least one of an amphoteric surfactant and a nonionic surfactant is added to at least one of the organic solvent and the aqueous medium.

What is claimed is: 
     
       1. A method of measuring the concentration of protease inhibitor in a sample comprising mixing the sample, a protease, calcium and a substrate, and then measuring the activity of the protease, wherein the calcium content is at least 0.15 μmol per 1 μg of the protease but no more than 100 μmol per 1 ml of the sample and wherein the substrate is used as a substrate solution containing an organic solvent, an aqueous medium and at least one amphoteric and/or nonionic surfactant. 
     
     
       2. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 1, wherein a buffer solution or water is used as the aqueous medium in preparing the substrate solution. 
     
     
       3. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 1, wherein the substrate solution is prepared by dissolving the substrate in an organic solvent, and diluting the solution with an aqueous medium, wherein the surfactant is added to at least one of the organic solvent and the aqueous medium. 
     
     
       4. A method of measuring the concentration of protease inhibitor in a sample as claimed in claim 1, wherein when a reaction solution is prepared by mixing the protease, the substrate, calcium and the sample, the reaction solution has a pH of 5 to 9, the protease concentration in the reaction solution is in the range of 5 to 250 mg/l, and the substrate concentration in the reaction solution is in the range of 0.5 to 25 mmol/l. 
     
     
       5. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 1, wherein the protease is trypsin. 
     
     
       6. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 5, wherein the sample is urine and the protease inhibitor is urinary trypsin inhibitor. 
     
     
       7. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 1, wherein the organic solvent used in preparing the substrate solution is dimethyl sulfoxide (DMSO). 
     
     
       8. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 1, wherein the substrate is expressed by the following Formula I:   protecting group-(amino acid residue).sub.n -p-nitroanilide;(Formula I)     wherein n is an integer between 1 and 5.   
     
     
       9. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 8, wherein the substrate is α-benzoyl-arginine-p-nitroanilide. 
     
     
       10. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 1, wherein the amphoteric surfactant is of the betaine type. 
     
     
       11. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 1, wherein the amphoteric surfactant is at least one of 3- (3-cholamidopropyl)dimethylammonio!-1-propanesulfonic acid and 3- (3-cholamidopropyl)dimethylammonio!-2-hydroxy-1-propanesulfonic acid. 
     
     
       12. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 1, wherein the nonionic surfactant is at least one selected from the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene (23) lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (10) octylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene alkyl ether, perfluoroalkyl polyoxyethylene ethanol, alkylester fluoride, polyethylene glycol mono-p-nonylphenylether, polyoxyethylene (30) octylphenyl ether, N,N-bis(3-D-gluconamidopropyl) deoxycholamide, n-octyl-β-d-thioglucoside, and sucrose monolaurate. 
     
     
       13. The method of measuring the concentration of protease inhibitor in a sample as claimed in claim 1, wherein the content of each component in the substrate solution is as follows: 1 to 50 mmol/l of substrate, 1 to 50% by weight of organic solvent, and 0.1 to 5% by weight of surfactant. 
     
     
       14. A kit for measuring the concentration of protease inhibitor in a sample comprising a protease, a substrate and calcium, wherein the calcium content is at least 0.15 μmol per 1 μg of the protease but no more than 100 μmol per 1 ml of the sample, and wherein the substrate is dissolved in a solution which contains an organic solvent, an aqueous medium and at least one amphoteric and/or nonionic surfactant. 
     
     
       15. The kit as claimed in claim 14, wherein a buffer solution or water is used as the aqueous medium in preparing the substrate solution. 
     
     
       16. The kit as claimed in claim 14, wherein the substrate solution is prepared by dissolving the substrate in an organic solvent, and diluting the solution with an aqueous medium, wherein the surfactant is added to at least one of the organic solvent and the aqueous medium. 
     
     
       17. The kit as claimed in claim 14, wherein when a reaction solution is prepared by mixing the protease, the substrate, calcium and the sample, the reaction solution has a pH of 5 to 9, the protease concentration in the reaction solution is in the range of 5 to 250 mg/l, and the substrate concentration in the reaction solution is in the range of 0.5 to 25 mmol/l. 
     
     
       18. The kit as claimed in claim 14, wherein the protease is trypsin. 
     
     
       19. The A kit as claimed in claim 18, wherein the sample is urine and the protease inhibitor is urinary trypsin inhibitor. 
     
     
       20. The kit as claimed in claim 14 comprising a buffer solution (R1), a protease solution (R2), and a substrate solution (R3), the ratio of R1, R2 and R3 by volume being in the range of R1:R2:R3=30 to 90:5 to 40:5 to 30, wherein (R1) is prepared so that it contains at least 0.15 μmol calcium per 1 μg of the protease but no more than 100 μmol calcium per 1 ml of the sample and wherein (R3) contains a substrate, an organic solvent and a surfactant, wherein the surfactant is at least one of an amphoteric surfactant and a nonionic surfactant. 
     
     
       21. The kit as claimed in claim 14, wherein the organic solvent used in preparing the substrate solution is dimethyl sulfoxide (DMSO). 
     
     
       22. The A kit as claimed in claim 14, wherein the substrate is expressed by the following Formula I:   protecting group-(amino acid residue).sub.n -p-nitroanilide;(Formula I)     wherein n is an integer between 1 and 5.   
     
     
       23. The kit as claimed in claim 22, wherein the substrate is α-benzoyl-arginine-p-nitroanilide. 
     
     
       24. The kit as claimed in claim 14, wherein the amphoteric surfactant is of the betaine type. 
     
     
       25. The kit as claimed in claim 14, wherein the amphoteric surfactant is at least one of 3- (3-cholamidopropyl)dimethylammonio!-1-propanesulfonic acid and 3- (3-cholamidopropyl)dimethylammonio!-2-hydroxy-1-propanesulfonic acid. 
     
     
       26. The kit as claimed in claim 14, wherein the nonionic surfactant is at least one selected from the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene (23) lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (10) octylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene alkyl ether, perfluoroalkyl polyoxyethylene ethanol, alkylester fluoride, polyethylene glycol mono-p-nonylphenylether, polyoxyethylene (30) octylphenyl ether, N,N-bis(3-D-gluconamidopropyl) deoxycholamide, n-octyl-β-d-thioglucoside, and sucrose monolaurate. 
     
     
       27. The kit as claimed in claim 14, wherein the content of each component in the substrate solution is as follows: 1 to 50 mmol/l of substrate, 1 to 50% by weight of organic solvent, and 0.1 to 5% by weight of surfactant. 
     
     
       28. A method of preparing a protease substrate solution comprising dissolving a protease substrate in an organic solvent to form a first solution and diluting the first solution with an aqueous medium, wherein at least one of an amphoteric surfactant and a nonionic surfactant is added to at least one of the organic solvent and the aqueous medium. 
     
     
       29. The method of dissolving a protease substrate as claimed in claim 28, wherein the aqueous medium is a buffer solution or water. 
     
     
       30. The method of dissolving a protease substrate as claimed in claim 28, wherein the organic solvent used in preparing the protease substrate solution is dimethyl sulfoxide (DMSO). 
     
     
       31. The method of dissolving a protease substrate as claimed in claim 28, wherein the protease substrate is expressed by the following Formula I:   protecting group-(amino acid residue).sub.n -p-nitroanilide;(Formula I)     wherein n is an integer between 1 and 5.   
     
     
       32. The method of dissolving a protease substrate as claimed in claim 31, wherein the substrate is α-benzoyl-arginine-p-nitroanilide. 
     
     
       33. The method of dissolving a protease substrate as claimed in claim 28, wherein the amphoteric surfactant is of the betaine type. 
     
     
       34. The method of dissolving a protease substrate as claimed in claim 28, wherein the amphoteric surfactant is at least one of 3- (3-cholamidopropyl)dimethylammonio!-1-propanesulfonic acid and 3- (3-cholamidopropyl)dimethylammonio!-2-hydroxyl-1-propanesulfonic acid. 
     
     
       35. The method of dissolving a protease substrate as claimed in claim 28, wherein the nonionic surfactant is at least one selected from the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene (23) lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (10) octylphenyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene alkyl ether, perfluoroalkyl polyoxyethylene ethanol, alkylester fluoride, polyethylene glycol mono-p-nonylphenylether, polyoxyethylene (30) octylphenyl ether, N,N-bis(3-D-gluconamidopropyl) deoxycholamide, n-octyl-p-d-thioglucoside, and sucrose monolaurate. 
     
     
       36. The method of dissolving a protease substrate as claimed in claim 28, wherein the content of each component in the protease substrate solution is as follows: 1 to 50 mmol/l of protease substrate, 1 to 50% by weight of organic solvent, and 0.1 to 5% by weight of surfactant.